Physiological and biochemical effects of triclocarban stress on freshwater algae
Triclocarban (TCC) is considered as a contaminant of emerging concern (CEC) and ranked in the top 10 CEC occurrence. TCC is a high-production-volume synthetic chemical used extensively in various personal care products. The heavy use and emission of TCC pose a serious threat to the environment. To evaluate its ecotoxicity, three freshwater algae were exposed to TCC at concentrations ranging from 0.500 to 16,000 mg/L to determine its growth inhibitory effects. Chlorophyll, malondialdehyde (MDA), antioxidant enzyme activities, and other indicators were used to analyze the mechanism underlying the toxic effect of TCC. With the 96-h median effective concentrations (EC50) of TCC ranging from 5.445 to 10.220 mg/L, the three types of algae exhibited obvious toxic responses, of which Chlorella vulgaris was the most sensitive. The calculated 96-h EC50 values of TCC were 8.474 mg/L, 9.100 mg/L, and 8.760 mg/L for C. vulgaris, Scenedesmus obliquus, and Chlorella pyrenoidosa, respectively. TCC decreased the chlorophyll content, increased the MDA content, decreased the peroxidase activity, and decreased superoxide dismutase activity in the three algae, indicating that the chemical can inhibit photosynthesis and destroy the activities of antioxidant enzymes in algae. Most of the indicators showed a positive correlation with the concentration of TCC.
KeywordsTriclocarban Green algae Chlorophyll Peroxidase Superoxide dismutase
The antimicrobial agents triclocarban (3,4,4′-trichlorocarbanilide; TCC) is high-production-volume chemicals that are widely used in a variety of personal care products (PCPs) . Its mass production and daily application have continued for more than 30 years, and a conservative estimate indicates that the world’s largest annual usage is over 750 × 103 t . Currently, TCC is among the top 10 most commonly detected organic wastewater compounds for frequency and concentration in the whole world . It has been detected in wastewater effluent in quantities up to micromolar concentrations (μg/L) in Asia, Europe, and North America, indicating widespread environmental contamination. This substance is highly lipophilic, can accumulate in various organisms, has been used for a long time, and poses potential threats to the environment and organisms. Although the detection of the instrument can quantify TCC concentration, it cannot measure directly the influences on test organisms. Due to the persistence and toxicity of TCC, its aquatic ecotoxicity has attracted wide attention . In recent years, its toxic effects, such as inhibitory effects on growth and development, endocrine disruption, and reproductive toxicity, have caused great concerns [5, 6, 7, 8, 9, 10, 11]. Several studies provided evidence that TCC interferes with biological receptors in vitro, including a stably transfected aryl hydrocarbon receptor (AhR)-, androgen receptor (AR)-, or estrogen receptor (ER)-responsive firefly luciferase reporter gene that responds to chemicals that can bind to and/or activate the respective receptor . Moreover, TCC could also be a significant concern when it coexists with TCS, whose hazard quotient was reported to be over 10 by Brausch and Rand .
Algae are at the bottom of the food chain in aquatic ecosystems. These organisms are sensitive to environmental pollution and are also an important segment in the accumulation of toxic substances, which can advance to consumers through the food chain. They are routinely employed as test organisms for ecotoxicological studies. Normally, the molecular, cellular, and physiological levels of algae change dramatically when they are under contamination stress. The examination of 139 water sources in 30 US states by the US Geological Survey  and that of the Zhujiang River, Yangtze River, Yellow River, and other water systems in China indicated that TCC was a highly accumulated pollutant. The maximum detected concentration in water is 338 ng/L that in the sediment reaches 2.723 μg/g, and the concentrations in water samples from wastewater treatment plants are as high as 0.4–50 μg/L [14, 15, 16]. TCC can also be detected in organisms, at levels ranging from 50 to 400 μg/kg in Cladophora, 50–300 μg/kg in freshwater snail, 14,000–80,000 μg/kg in bream bile, 0.75–10 μg/kg in pelagic fish plasma, 9.0 μg/kg in killer whale plasma, 0.4–38.0 ng/g in the plasma of lactating mothers, and 0.022–0.95 ng/g in the breast milk [17, 18]. Therefore, the ecological toxicity of TCC requires attention.
This study aimed to treat three typical freshwater algae with different concentrations of TCC and then analyze the growth inhibition, photosynthesis, and antioxidant capacity of algae to evaluate the possible toxic effect of TCC on algae and its underlying mechanism to lay a foundation for the control of environmental pollution by TCC and for biological toxicology research.
2 Materials and methods
The TCC standard was obtained from J&K Scientific Ltd (Beijing, China) with a purity of 97%. The HPLC-grade reagents including methanol, acetonitrile, ethyl acetate, hexane, dichloromethane, formic acid, and acetic acid were supplied by Merck (Germany), CNW Technologies (China), and Tedia (USA). Glass fiber filters (GF/F, pore size 0.7 μm) were obtained from Whatman (Maidstone, UK). Oasis HLB cartridges (200 mg, 6 mL) were from Waters (Milford, USA). HPLC-grade water was from a Milli-Q water purification system (Millipore, Watford).
2.2 Test organisms
To create a representative subtropical community, we used three species of algae: Chlorella vulgaris, Scenedesmus obliquus, and Chlorella pyrenoidosa, which were provided by the Institute of Hydrobiology, Chinese Academy of Sciences. The algae were laboratory cultured in BG11 medium at 23 ± 1 °C and a light intensity of 4000 lux(provided by 40 W white fluorescent lamps, Philips F40 T12/DX) with a dark/light cycle of 14 h/10 h. The preparation of BG11 medium can be found in a previous study . The biomass was measured by microscopic counting and optical density (OD) at 690 nm at 24-h intervals during 120 h of culture. Growth curves were plotted to establish the relationship between the cell density and OD690 values for the different algae using a linear regression equation.
2.3 Toxic test of TCC on algae
2.3.1 Exposure strategies
In the exposure experiment, the algae at the logarithmic growth phase were collected, washed, and then diluted by freshwater to the initial concentration of 2 × 105 cells mL−1. The three species of algae were cultured with the final concentrations of TCC of 0.5, 1, 2, 4, 8, and 16 mg/L. After 96 h of culture, the biomass, chlorophyll, malondialdehyde (MDA), peroxidase (POD), superoxide dismutase (SOD), and other indicators were measured to evaluate the corresponding toxic effects, with the biomass analyzed once every 24 h . Three replicates were set for each concentration group.
2.3.2 Growth inhibition of algae and calculation of the 96-h median effective concentration (EC50)
2.3.3 Determination of the chlorophyll content in algae
2.3.4 Determination of the algal MDA content
2.3.5 Determination of algal SOD and POD activities
SOD activity was assayed at 560 nm by the nitro-blue-tetrazolium (NBT) photoreduction method . One unit of SOD activity was defined as the amount of enzyme required to cause 50% inhibition of the reduction in NBT as monitored at 560 nm by a spectrophotometer (722-type, Shanghai Instrument Factory, China). Scavenging of O2− by SOD prevents the accumulation of intermediates, which is used for the determination of the enzyme activity. POD activity was determined at 470 nm using the guaiacol method . The reaction mixture (3 mL) contained 50 mM PBS (Ph 5.5), 20 mM guaiacol, 1 mL 30% (w/v) H2O2, and 0.1 mL enzyme extract.
2.4 Statistical methods
All experiments were performed at least in triplicate. The quantitative data were expressed as mean and standard deviation/standard error. If the homoscedasticity assumptions of the data were satisfied, least significant difference (LSD) tests were further conducted to analyze the statistical significance among individual treatments using SPSS v25 (IBM, Armonk, NK, USA). P < 0.05 was considered statistically significant, and P < 0.01 was considered highly significant.
3.1 Establishment of a spectrophotometric method for algal biomass
3.2 Inhibitory effects of TCC on the growth of the three species of algae
3.3 Effects of TCC on the chlorophyll content in the three algae
3.4 Effects of TCC on the MDA content in the three algae
MDA is the final decomposition product of membrane lipid peroxidation, and its content reflects the degree of stress damage to algae. The TCC concentrations in the range tested in this study caused an increase in the MDA content, indicating that the algae experienced stress, leading to cell membrane damage and increased cell membrane permeability. Using the TCC effect on S. obliquus as an example, the MDA content in the control group was of 0.068 μmol/g, and that in the treated groups was 0.060, 0.093, 0.105, 0.125, 0.154, and 0.141 μmol/g, which was up to 2.35 times of that in the control group.
3.5 Effects of TCC on the POD activity in the three algae
3.6 Effects of TCC on the SOD activity in the three algae
POD is the enzyme responsible for the decomposition of peroxides, while SOD is the enzyme responsible for scavenging oxygen free radicals, and both are key enzymes allowing algae to resist oxidative damage and remove metabolic wastes, which is very important for the growth and metabolism of algae. TCC was shown to significantly damage both activities. Taking the effect of TCC on the POD activity in C. pyrenoidosa as an example, the activity in the control group was 32 mg/g/min and that in the exposed groups was 29, 25, 20, 16, 13, and 9 mg/g/min. The lowest activity in the exposed groups was 28.13% of that in the control group. Taking the effect of TCC on the SOD activity in C. pyrenoidosa as an example, the activity in the control group was 178 U/g·fresh weight (Fw) and that in the exposed groups was 160, 143, 118, 88, 56, and 27 U/g Fw. The lowest activity in the exposed groups was 15.17% of that in the control group.
The effects of TCC differed among the organisms that were introduced to the microcosms. In these microcosms, both phytoplanktonic and periphytonic chlorophyll-a content decreased as the concentrations of TCC in the sediment increased. Algae are known to be particularly sensitive to TCC, with growth inhibition typically being recorded at EC50 values of 0.53 μg/L–430 μg/L, depending on the test conditions and species [23, 24]. In this study, TCC caused a significant decrease in the chlorophyll content in the algae. The 96-h EC50 values of TCC for C. pyrenoidosa, C. vulgaris, and S. obliquus were between 5.445 and 10.22 mg/L. Although the TCC content in most of the current water systems has not yet reached the 96-h EC50 values for the algae, the lowest-concentration groups of the algae (0.5 mg/L) also showed some toxicity effects, indicating that the monitoring and control of TCC should be strengthened.
Among the indexes analyzed in this study, chlorophyll was the executor of photosynthesis, and its content directly reflects the growth and reproduction ability of algae. Taking the effect of TCC on C. vulgaris as an example, the concentration of chlorophyll-a in the control group was 0.764 μg/L, while that in each group was 0.623, 0.496, 0.391, 0.295, 0.227, and 0.130 μg/L, respectively. The lowest group was only 20.86% of that in the control group, the effect was very obvious, and the experiment of other algae showed similar characteristics.
TCC and its transformation products (4,4′-dichlorocarbilide (DCC), 1-(3-chlorophenyl)-3-phenylurea (MCC) and carbanilide (NCC), 2′OH-TCC, 3′OH-TCC) were detected in the environmental matrices. TCC is an antimicrobial agent and also emerging endocrine disruptor that can cause immune dysfunction and affect human reproductive outcomes. Furthermore, TCC alters the expression of proteins related to binding and metabolism, skeletal muscle development and function, and nervous system development and immune response. TCC has potential health risks in wildlife and humans. Several animal studies illustrate that it can cause various adverse effects, which can be monitored by antioxidant biomarkers (CAT, GST, and LPO) . The first mechanism is a possible increase in intracellular oxidative stress, which could arise from a pathological increase in the production of oxyradicals (mainly H2O2, which is produced in mitochondria by dismutation of O2−) through the cellular mitochondrial enzymatic complex chain . Alternatively, TCC may act together as a DNA adduct and/or DNA intercalant to directly exert genotoxic effects .
Numerous uncertainties remain including the evaluation of antioxidant enzymes, the possible interference with hormone metabolism, and the role played by the metabolites of TCC. It is necessary to fully understand the biological toxic effect of TCC on this sentinel organism. As these questions continue to be addressed, more definitive and comprehensive aquatic risk assessments should be developed to raise greater public awareness about the potential risk of TCC released into aquatic environments.
TCC showed a significant acute toxicity to the freshwater algae, significantly inhibiting their growth, destroying their photosynthetic ability, and impairing their antioxidant function. Because this study was a short-term exposure study, taking into account a long-term cumulative effect of TCC in the environment, it is necessary that TCC should be listed as an important environmental pollutant of great concern, and its content should be tested in each major river system. Furthermore, this paper provides the basic information regarding the physiological and biochemical effects of TCC stress on freshwater alga, and further systematic toxicology research should be carried out on model organisms at all levels to understand toxicity mechanisms.
The authors of this study express their gratitude to the National Natural Science Foundation of China (31600257) and Public welfare project of Zhejiang provincial Science and Technology Department (2016C32022) for financial support of this study.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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